The possibility of developing an antifertility vaccine for male mammals based on active immunization against Luteinizing Hormone Releasing Hormone (LHRH) is currently under investigation in several laboratories. These studies are based on the findings that antibodies against LHRH prevent gonadotropin release. Prevention of gonadotropin release causes regression of Leydig cells and suppression of testosterone production and spermatogenesis with subsequent infertility. See e.g. Fraser et al., "Effect of Active Immunization to Luteinizing Hormone Releasing Hormone on Serum and Pituitary Gonadotropins, Tests and Accessory Sex Organs in Male Rat", J. Endocrinol., 63:399-405 (1974); Fraser et al., "LHRH Antibodies: Their Use in the Study of Hypothalamic LHRH and Testicular LHRH-like Material, and Possible Contraceptive Applications", In: Sandlet, ed Progress Toward a Male Contraceptive. New York, John Wiley, (1982) pp. 41-78.
Production of anti-LHRH antibodies would also be useful to treat certain forms of cancer such as prostate cancer where such therapy could replace orchiectomy or LHRH analogue treatment.
Recurrent or metastatic cancer of the prostate is a major cause of cancer mortality in the United States with more than 25,000 deaths occurring annually because of this malignancy. Silverberg and Lubera, "Cancer Statistics", CA 38:1-19 (1988). It is the most common malignancy in men older than 70. Carcinoma within the prostate is a common finding at autopsy, being found in 20-40% of men between 70-79 years of age. Unfortunately, approximately one third of all prostate cancers are diagnosed only after the patient has clinically apparent disseminated disease with bone or visceral metastases outside the pelvis.
For patients with disseminated disease at the time of diagnosis the classical treatment is surgical orchiectomy. Huggins and Hodges, "Studies of Prostatic Cancer. Effect of Castration, Estrogen, and Androgen Injection of Serum Phosphatases in Metastatic Carcinoma of the Prostate", Cancer Res., 1:293-297 ( 1941 ). This procedure decreases serum testosterone levels by 95% and causes objective tumor regression in approximately 40% of cases and disease stabilization in an additional 40% of cases. Alternative strategies to surgical orchiectomy include treatment with diethylstilbestrol (DES) or with LHRH analogues. These strategies also seem to work by decreasing serum testosterone levels, and have similar response rates to those seen with orchiectomy. Results of first line single agent hormonal manipulations are favorable but average response durations are less than one year and actuarial survival less than 2 years. Seftel et al., "Hormonal Therapy for Advanced Prostatic Carcinoma", J. Surg. Onc. Suppl., 1: 14-20 ( 1989 ).
Recent results have suggested that the addition of an anti-androgen to initial orchiectomy or an LHRH antagonist may improve response rates. Using combined androgen blockade in patients with stage D2 prostate cancer, Labtie initially reported a 90% survival rate at 2 years. When this therapy was tested in a Canadian double-blinded randomized trial, the results still statistically significantly favored combined therapy although the improvement in survival was less striking (52% survival at 18 months with simple orchiectomy versus 66% survival with combined therapy). Beland et al., "Total Androgen Blockade for Metastatic Cancer of the Prostate", Am. J. Clin. Onc., 11 (Suppl. 2):S18714 S190 (1988) This modest but statistically significant improvement in survival provided by combined therapy was also seen in an American randomized study. Thus the optimal therapy for cancer of the prostate at this time seems to be a combination of orchiectomy and therapy that blocks peripheral androgen action.
Current therapies for the suppression of androgen production are not universally acceptable to all patients. Orchiectomy is psychologically unacceptable to many patients. The user of LHRH analogues is dependent on repetitive application of the analogue and involves long term inconvenience and expense. Use of estrogens such as DES is generally considered inferior to the other two treatment modalities due to cardiovascular complications and side effects such as gynecomastia.
Immunization against LHRH has been proposed as an alternative anti-neoplastic strategy for sex steroid dependent tumors. Raydin and Jordan, "Active Immunization to Luteinizing Hormone to Inhibit the Induction of Mammary Tumors in the Rat", Life Sci., 43:117-123 (1988). This strategy has been shown to be effective in animal models, and depends on the immunoneutralization of LHRH after active immunization of the host. This blockade of LHRH causes decreased production of gonadotropins and then secondarily decreased sex steroid production by the ovaries or testes. It would be of great significance in the treatment of this disease to have a LHRH vaccine capable of rapidly stimulating production of high titers of anti-LHRH antibodies.
The availability of synthetic LHRH has provided anti-LHRH antisera, needed for development of radioimmunoassays and immunocytochemistry. Unfortunately, the use of synthetic LHRH in humans to induce production of anti-LHRH antibodies is precluded by the necessity of using adjuvants not suitable for use in humans in order to achieve an effective titer within an acceptable time period. In the majority of the reported experiments, Freund's complete adjuvant (FCA) was used as an immune enhancer to obtain anti-LHRH antisera. Chappel et al., "Active Immunization of Male Rhesus Monkeys Against Luteinizing Hormone Releasing Hormone", Biol. Reprod., 22:333-342 (1974); Fraser et al., "Preparation of Antisera to Luteinizing Hormone Releasing Factor", J. Endocrinol , 61:756-769 (1974); and Awoniyi et al., "Changes in Testicular Morphology in Boars Actively Immunized Against Gonadotropin Hormone-Releasing Hormone", J. Androl., 9:160-171 (1988).
In developing vaccines for man, only materials permitted for use in humans can be utilized. This criterion eliminates FCA, the most potent immune enhancer. Thus other methods of inducing highly effective anti-LHRH antibody in the shortest possible time are necessary to enable anti-LHRH production in man. Steblay, "Glomerulonephritis Induced in Sheep By Injections of Heterologous Glomerular Basement Membrane and Freund's Complete Adjuvant", Exp. Med., 116:253-172 (1962); Thau et al., "Effects of Immunization with the 8-Subunit of Ovine Luteinizing Hormone on Corpus Luteum Function in the Rhesus Monkey", Fertil. Steril., 31:200-204 (1979); and Dalsgaard, "Adjuvants", Vet. Immunol. Immunopathol., 17:145-152 (1987).
The studies described below were designed to examine the ability of LHRH conjugated to tetanus toxoid (TT) either at the N-terminal, C-terminal, or mid-section of the LHRH to induce biologically effective antibodies in the shortest possible time.
It has now been shown that conjugating a protein carrier to LHRH enhances immunogenicity in a site specific manner. Carriers such as TT and purified protein derivative (PPD, derived from tuberculin) which are suitable for use in man and have been shown to stimulate the immune response are useful as the protein carrier. Fraser et al., (1982); Shastri et al., (1981); Talwar, "Immunobiology of Gonadotropin-Releasing Hormone", J. Steroid Biochem., 23: 795-800 ( 1985 ); and Ellouz et al., "Minimal Structural Requirements for Adjuvant Activity of Bacterial Peptidoglycan Derivative", Biochem. and Biophys. Res. Comm.,59:1317-1325 (1974).